This invention relates to a process for preparing biodegradable microspheres and/or nanospheres using an oil-in-water process, which microspheres and nanospheres can be used for the controlled release of bioactive peptides.
A variety of techniques are described in the literature for the preparation of polymer microspheres for the sustained release of bioactive peptides. Among the different techniques such as spray drying, spray congealing, coacervation, solvent evaporation etc., solvent evaporation is simplest to scale-up industrially (for a recent review see protein delivery from biodegradable microspheres, by J. L. Cleland in Protein Delivery edited by L. Sanders and W. Hendren, Plenum Press, N.Y. 1997). Solvent evaporation is usually practiced by dissolving or suspending an active ingredient in a polymer solution, which is further dispersed in the form of droplets in a suitable medium containing surfactants capable of stabilizing the droplets, and the polymer droplets are hardened by evaporation of the solvent. When the polymer is dissolved in an organic medium and then emulsified in water, the process is called oil-in-water process (O/W). Water soluble peptides cannot be encapsulated by the O/W process, due to the partition of the water soluble peptides into the aqueous medium, resulting in low encapsulation efficiency. Higher encapsulation efficiencies were achieved by a more complex double emulsion water-in-oil-in-water (W/O/W) process (U.S. Pat. No. 5,271,945) or by using an oil-in-oil (O/O) process (EP 0330180 B1). The main drawback of the latter process is the use of different organic solvents, first to solubilize the polymer, and then to wash the polymer microspheres free of the oil in which they are formed. Therefore, the simple O/W emulsion solvent evaporation process is the most attractive, provided higher encapsulation efficiency can be achieved, since only one organic solvent is involved, and the residual organic solvent can be removed by vacuum drying.
The main hurdle to achieving higher encapsulation efficiency of the peptides is their water solubility. Solubility of peptides depends on the nature of the counter-ion. The aqueous solubility of a peptide is considerably reduced when the peptide is present as a free base, due to intermolecular interactions. One method of enhancing the encapsulation efficiency of the peptides in an O/W process according to the present invention, is by using a peptide as a free base adsorbed onto a bioresorbable inorganic matrix, such as hydroxyapatite, Calcium monohydrogen phosphate, zinc hydroxide, alum etc. In the case of encapsulation of LHRH agonists such as tryptorelin, leuprolin, goserlin, busrelin, etc., the presence of calcium phosphate in the micropheres may not only serve to stabilize the neutralized peptide but also act as a calcium supplement, since one of the biggest concerns of continuous therapy using LHRH agonists is loss of bone density. This method of encapsulation is most suited when the peptide loading in excess of 5-6% is not desired. In the case of high peptide loading, a heterogeneous distribution of the drug particles, even if they were stabilized by adsorption onto a solid matrix or not, inside the microspheres leads to non-predictable release profiles.
In cases where higher drug loading as well as predictable release profiles are desired, a second method of reducing the aqueous solubility of the drug, without sacrificing its potency, is by simply forming reversible water insoluble salts of mono-functional or multi-functional detergents and/or polymers or a combination of both, as exemplified by Schally et al. in U.S. Pat. No. 4,010,125. The aqueous solubility of the peptides can be considerably reduced by forming salts of mono-functional detergents such as sodium dodecyl sulfate, or of multi-functional anionic species such as pamoate, tannate, alginate, carboxymethyl cellulose, leading to the precipitation of the water insoluble peptide salt. Among the water insoluble salts, some exhibit good solubility in common organic solvents. U.S. Pat. No. 5,672,659 describes compositions formed between anionic carboxylate functionalized polyesters and cationic peptides. These compositions as well as those formed with certain anionic detergents such as dioctylsulfosuccinate are found to exhibit good solubility in organic solvents such as dichloromethane (DCM), chloroform, acetonitrile, ethyl acetate, and the like.
During the water based encapsulation of the peptide, either as a free base adsorbed on to solid matrix or as water insoluble but organic solvent soluble salt, the pH of the aqueous medium can dramatically increase the water solubility, by affecting the equilibrium between the complexed and uncomplexed state. If the pH is not maintained at 7 the equilibrium may shift, favoring the solubilization of the peptide, leading to poor encapsulation efficiency.
It is therefore the object of the present invention to provide polymer microspheres and/or nanospheres prepared by a simple O/W method, where the encapsulation efficiency achieved can be greater than 85%.